1. Field of the Invention
The present invention describes nucleic acid sequences for eukaryotic genes encoding ∈ lycopene ∈-cyclase (also known as ∈-cyclase and ∈ lycopene cyclase), isopentenyl pyrophosphate isomerase (IPP) and xcex2-carotene hydroxylase as well as vectors containing the same and hosts transformed with said vectors. The present invention also provides methods for augmenting the accumulation of carotenoids, changing the composition of the carotenoids, and producing novel and rare carotenoids. The present invention provides methods for controlling the ratio or relative amounts of various carotenoids in a host. The invention also relates to modified lycopene ∈-cyclase, IPP isomerase and xcex2-carotene hydroxylase. Additionally, the present invention provides a method for screening for genes and cDNAs encoding enzymes of carotenoid biosynthesis and metabolism.
1. Background of the Invention
Carotenoid pigments with cyclic endgroups are essential components of the photosynthetic apparatus in oxygenic photosynthetic organisms (e.g., cyanobacteria, algae and plants; Goodwin, 1980). The symmetrical bicyclic yellow carotenoid pigment xcex2-carotene (or, in rare cases, the asymmetrical bicyclic xcex1-carotene) is intimately associated with the photosynthetic reaction centers and plays a vital role in protecting against potentially lethal photooxidative damage (Koyama, 1991). xcex2-carotene and other carotenoids derived from it or from xcex1-carotene also serve as light-harvesting pigments (Siefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the light-harvesting antenna (Demmig-Adams and Adams, 1992), provide substrate for the biosynthesis of the plant growth regulator abscisic acid (Rock and Zeevaart, 1991; Parry and Horgan, 1991), and are precursors of vitamin A in human and animal diets (Krinsky, 1987). Plants also exploit carotenoids as coloring agents in flowers and fruits to attract pollinators and agents of seed dispersal (Goodwin, 1980). The color provided by carotenoids is also of agronomic value in a number of important crops. Carotenoids are currently harvested from a variety of organisms, including plants, algae, yeasts, cyanobacteria and bacteria, for use as pigments in food and feed.
The probable pathway for formation of cyclic carotenoids in plants, algae and cyanobacteria is illustrated in FIG. 1. Two types of cyclic endgroups or rings are commonly found in higher plant carotenoids, these are referred to as the xcex2 (beta) and ∈ (epsilon) rings (FIG. 3). The precursor acyclic endgroup (no ring structure) is referred to as the xcexa8 (psi) endgroup. The xcex2 and ∈ endgroups differ only in the position of the double bond in the ring. Carotenoids with two xcex2 rings are ubiquitous, and those with one xcex2 and one ∈ ring are common, but carotenoids with two ∈ rings are uncommon. xcex2-carotene (FIG. 1) has two xcex2-endgroups and is a symmetrical compound that is the precursor of a number of other important plant carotenoids such as zeaxanthin and violaxanthin (FIG. 2).
Genes encoding enzymes of carotenoid biosynthesis have previously been isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol. Gen. Genet. 216, 254-268; Misawa et al., 1990, J. Bacteriol., 172, 6704-12), fungi (Schmidhauser et al., 1990, Mol. Cell. Biol. 10, 5064-70), cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86; Cunningham et al., 1994) and higher plants (Bartley et al., Proc. Natl. Acad. Sci USA 88, 6532-36; Martinez-Ferez and Vioque, 1992, Plant Mol. Biol. 18, 981-83). Many of the isolated enzymes show a great diversity in structure, function and inhibitory properties between sources. For example, phytoene desaturases from the cyanobacterium Synechococcus and from higher plants and green algae carry out a two-step desaturation to yield xcex6-carotene as a reaction product. In plants and cyanobacteria a second enzyme (xcex6-carotene desaturase), similar in amino acid sequence to the phytoene desaturase, catalyzes two additional desaturations to yield lycopene. In contrast, a single desaturase enzyme from Erwinia herbicola and from other bacteria introduces all four double bonds required to form lycopene. The Erwinia and other bacterial desaturases bear little amino acid sequence similarity to the plant and cyanobacterial desaturase enzymes, and are thought to be of unrelated ancestry. Therefore, even with a gene in hand from one source, it may be difficult to identify a gene encoding an enzyme of similar function in another organism. In particular, the sequence similarity between certain of the prokaryotic and eukaryotic genes encoding enzymes of carotenoid biosynthesis is quite low.
Further, the mechanism of gene expression in prokaryotes and eukaryotes appears to differ sufficiently such that one cannot expect that an isolated eukaryotic gene will be properly expressed in a prokaryotic host.
The difficulties in isolating genes encoding enzymes with similar functions is exemplified by recent efforts to isolate the gene encoding the enzyme that catalyzes the formation of xcex2-carotene from the acyclic precursor lycopene. Although a gene encoding an enzyme with this function had been isolated from a bacterium, it had not been isolated from any photosynthetic procaryote or from any eukaryotic organism. The isolation and characterization of the enzyme catalyzing formation of xcex2-carotene in the cyanobacterium Synechococcus PCC7942 was described by the present inventors and others (Cunningham et al., 1993 and 1994). The amino acid sequence similarity of the cyanobacterial enzyme to the various bacterial lycopene xcex2-cyclases is so low (ca. 18-25% overall; Cunningham et al., 1994) that there is much uncertainty as to whether they share a common ancestry or, instead, represent an example of convergent evolution.
The need remains for the isolation of eukaryotic and prokaryotic genes and cDNAs encoding polypeptides involved in the carotenoid biosynthetic pathway, including those encoding a lycopene ∈-cyclase, IPP isomerase and xcex2-carotene hydroxylase. There remains a need for methods to enhance the production of carotenoids, to alter the composition of carotenoids, and to reduce or eliminate carotenoid production. There also remains a need in the art for methods for screening for genes and cDNAs encoding enzymes of carotenoid biosynthesis and metabolism.
Accordingly, a first object of this invention is to provide purified and/or isolated nucleic acids which encode enzymes involved in carotenoid biosynthesis; in particular, lycopene ∈-cyclase, IPP isomerase and xcex2-carotene hydroxylase.
A second object of this invention is to provide purified and/or isolated nucleic acids which encode enzymes which produce novel or uncommon carotenoids.
A third object of the present invention is to provide vectors containing said genes.
A fourth object of the present invention is to provide hosts transformed with said vectors.
Another object of the present invention is to provide hosts which accumulate novel or uncommon carotenoids or which accumulate greater amounts of specific or total carotenoids.
Another object of the present invention is to provide hosts with inhibited and/or altered carotenoid production.
Another object of this invention is to secure the expression of eukaryotic carotenoid-related genes in a recombinant prokaryotic host.
Yet another object of the present invention is to provide a method for screening for eukaryotic and prokaryotic genes and cDNAs which encode enzymes involved in carotenoid biosynthesis and metabolism.
An additional object of the invention is to provide a method for manipulating carotenoid biosynthesis in photosynthetic organisms by inhibiting the synthesis of certain enzymatic products to cause accumulation of precursor compounds.
Another object of the invention is to provide modified lycopene ∈-cyclase, IPP isomerase and xcex2carotene hydroxylase.
These and other objects of the present invention have been realized by the present inventors as described below.
A subject of the present invention is an isolated and/or purified nucleic acid sequence which encodes for a protein having lycopene ∈-cyclase, IPP isomerase or xcex2-carotene hydroxylase enzyme activity and having the amino acid sequence of SEQ ID NOS:2, 4, 14-21, 23 or 25-27.
The invention also includes vectors which comprise any of the nucleic acid sequences listed above, and host cells transformed with such vectors.
Another subject of the present invention is a method of producing or enhancing the production of a carotenoid in a host cell, comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein having lycopene ∈-cyclase, IPP isomerase or xcex2-carotene hydroxylase enzyme activity, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence to produce the protein.
Yet another subject of the present invention is a method of modifying the production of carotenoids in a host cell, the method comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence which produces an RNA and/or encodes for a protein which modifies lycopene ∈-cyclase, IPP isomerase or xcex2-carotene hydroxylase enzyme activity, relative to an untransformed host cell, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence in the host cell to modify the production of the carotenoids in the host cell, relative to the untransformed host cell.
The present invention also includes a method of expressing, in a host cell, a heterologous nucleic acid sequence which encodes for a protein having lycopene ∈-cyclase, IPP isomerase or xcex2-carotene hydroxylase enzyme activity, the method comprising inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence.
Also included is a method of expressing, in a host cell, a heterologous nucleic acid sequence which encodes for a protein which modifies lycopene ∈-cyclase, IPP isomerase or xcex2-carotene hydroxylase enzyme activity in the host cell, relative to an untransformed host cell, the method comprising inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence.
Another subject of the present invention is a method for screening for genes and cDNAs which encode enzymes involved in carotenoid biosynthesis and metabolism.